Biology Reference
In-Depth Information
10.3
IMAGING GTP ISLANDS IN MICROTUBULES ASSEMBLED
IN VITRO
In vitro
techniques may also be used to visualize GTP-tubulin regions in microtu-
bules. These approaches allow GTP island formation in the absence of MAPs or
of insoluble cellular structures after detergent extraction. One of the following pro-
tocols also makes use of the GTP analogue GMPCPP, which locks the conformation
of tubulin in a GTP-like state. GMPCPP is a slowly hydrolyzable molecule used
formerly by Hyman and colleagues (
Hyman et al., 1992
) who showed that GTP
hydrolysis is essential for depolymerization of microtubules and thus for dynamic
instability.
10.3.1
Microtubules assembled in suspension with GTP or GMPCPP
10.3.1.1
Separate preparation of GMPCPP and GTP microtubules
Unlabeled and fluorescent tubulins (see
Section 3.2
) are used to polymerize GMPCPP
and GTP microtubules separately. The ratio between fluorescent tubulin and nonfluor-
escent tubulin is 1:10. Tubulin diluted in PEM supplemented with 10 mMDTT is used
at 2.5 g/L (final concentration) and incubated on ice with 1 mM GTP or 1 mM
GMPCPP for 5 min. Both mixes are polymerized for 30-45 min at 37
C. After poly-
merization, microtubules are diluted at least 50- to 100-fold in warm PEM-T.
10.3.1.2
Mixed GMPCPP and GTP microtubules and staining with MB11
antibody
Microtubules polymerized separately in the presence of each nucleotide are spun
down at 30
C for 10 min at 20,000
g
to remove unpolymerized tubulin. Both
pellets are resuspended separately in a small volume of PEM-T and then one volume
of GMPCPP microtubules is mixed with one volume of GTP microtubules. The
MB11 antibody is added to the mixture, which is incubated for 15 min at 37
C.
Microtubules are centrifuged for 15 min at 20,000
g
at 30
C and washed once
in PEM-T. They are spun down again; the pellet is resuspended in PEM-T containing
the secondary antibody and incubated 15 min at 37
C.
After final pelleting for 10 min at 20,000
g
and resuspension in PEM-T supple-
mented with antifading agents, a drop of the microtubule suspension is directly ob-
served without fixation at room temperature, with a fluorescence microscope.
Tips
- Topipet polymerizedmicrotubules, use large pipette tips or cut the extremity
of the tip so that pipetting does not break polymerized microtubules.
- A small amount of Taxol (1
m
M) allows stabilization of the microtubules
without changing their conformation as described above. Adding large
amounts of Taxol will induce changes inmicrotubule conformation and such
microtubules will be stained all along the lattice by the MB11 antibody.
